PREPARATION AND CHARACTERIZATION OF COBALT(III)-GLUTAMINE AND CHROMIUM(III)-GLUTAMINE SYNTHETASE DERIVATIVES

Stoichiometric incorporation of Co(III) into the n1 metal ion sites of Escherichia coli glutamine synthetase (unadenylylated) was carried out by H2O2 oxidation of enzyme-bound Co(II). This enzyme derivative bound 1 mol of Mn(II)/subunit to the n2 metal-nucleotide sites with a Kd value of 0.67 .+-. 0.11 mM. The Co(III)-enzyme was catalytically inactive (< 0.1%) as judged by the .gamma.-glutamyl transferase assay. EPR studies of Mn(II)-nucleotide complexes bound to the Co(III)-enzyme revealed differences in the environment of Mn(II) with ATP or ADP in the presence of the other substrates L-glutamate and NH4+. The ATP analogs AMPPCP [adenosine methylene diphosphonate] and AMPPNP [adenylyl imidodiphosphate] produced different enzyme-bound Mn(II) spectra than ATP. L-Methionine sulfoximine, a transition-state analog for glutamine synthetase, gave an anisotropic Mn(II) spectrum with various nucleotides but the Mn(II) spectrum of Co(III)-enzyme-Mn(II) was quite distinct from the one that the sulfoximine produces when Mn(II) is in the n1 metal ion site. The Mn(II) environment at the n2 site is distinct from that previously observed for Mn(II) bound at the n1 site. The paramagnetic Cr(III)-enzyme derivative (n1 sites) was prepared by air oxidation of Cr(II)-enzyme. EPR studies were used to calculate a metal-metal distance between the n1 and N2 sites using Mn(II)-nucleotide complexes bound to the n2 site. The metal-metal distance of 7 .+-. 2 .ANG. is in good agreement with distances previously obtained for Mn(II)-enzyme-Cr(III)-nucleotide complexes (Mn(II) in n1 and Cr(III)-nucleotide in n2).